Short peptides which selectively modulate the activity of serine/threonine kinases

ABSTRACT

Disclosed are peptides which are peptide derivatives of the HJ loop of a serine/threonine kinase. The peptides can modulate the activity of the serine/threonine kinase. Also disclosed are methods of modulating the activity of a serine/threonine kinase in a subject by administering one of the peptides of the present invention.

BACKGROUND OF THE INVENTION

Serine/threonine kinases are a member of the eukaryotic protein kinasesuperfamily. Enzymes of this class specifically phosphorylate serine orthreonine residues of intracellular proteins and are important inmediating signal transduction in multicellular organisms. Manyserine/threonine kinases occur as intracellular proteins which take partin signal transduction within the cell, including signal transduction tothe nucleus and the activation of other proteins. Other serine/threoninekinases, such as G protein-coupled receptor kinases, are found in cellmembranes and participate in trans-membrane signalling.

As such, phosphorylation of serine or threonine by serine/threoninekinases is an important mechanism for regulating intracellular events inresponse to environmental changes. A wide variety of cellular events areregulated by serine/threonine kinases. A few examples include theability of cells to enter and/or complete mitosis, cellularproliferation, cellular differentiation, the control of fat metabolism,immune responses, inflammatory responses and the control of glycogenmetebolism.

Thus, agents which can modulate (increase or decrease) the activity ofserine/threonine kinases have great potential for the treatment of awide variety of diseases and conditions such as cancer, obesity,autoimmune disorders, inflammation and Type II diabetes.

SUMMARY OF THE INVENTION

It has now been found that short peptides which are derivatives of theHJ loop of a serine/theonine kinase can significantly affect theactivities of cells expressing the serine/threonine kinase (“HJ loop” isdefined hereinbelow) For example, the peptide drivatives of the HJ loopof Raf and Polo inhibit the proliferation of bovine aortic cells and thetransformed mouse cell lines MS1 and/or SVR cells in vitro atconcentrations as low as 10 μM (Example 2). Based on the aforementioneddiscoveries, novel peptides are disclosed herein which are peptidederivatives of the HJ loop of serine/threonine kinases. Also disclosedare methods of identifying a peptide derivative of an HJ loop of aserine/threonine kinase which modulates the activity of saidserine/threonine kinase. Methods of modulating the activity of aserine/threonine kinase in a subject are also disclosed.

One embodiment of the present invention is a novel peptide which is apeptide derivative of the HJ loop of a serine/threonine kinase. Thepeptide comprises between about five and about twenty amino acidresidues or amino acid residue analogs and modulates the activity of theserine/threonine kinase. The N-terminus and/or C-terminus of the peptidecan be substituted or unsubstituted. The peptide can be linear orcyclic.

Another embodiment of the present invention is a method of modulatingthe activity of a serine/threonine kinase in a subject. The methodcomprises administering a therapeutically effective amount of a peptidewhich is a derivative of an HJ loop of said serine/threonine kinase, asdescribed above.

Yet another embodiment of the present invention is a method ofidentifying a peptide which modulates the activity of a serine/threoninekinase. The method comprises providing a “test peptide” which has fromabout five to about twenty amino acids or amino acid analogs and whichis a peptide derivative of the HJ loop of said serine/threonine kinase.The test peptide is incubated with cells having a cellular activity orfunction under the control of said serine/threonine kinase underconditions suitable for assessing the activity of the serine/threoninekinase. The activity of the serine/threonine kinase is assessed andcompared with cells of the same cell type grown under the sameconditions in the absence of the test peptide. A greater or lesseractivity compared with cells grown in the absence of the test peptideindicates that the test peptide modulates activity of theserine/threonine kinase.

The peptides of the present invention can be used in the treatment of awide variety of diseases caused by overactivity and underactivity of aSTK. Examples include, but are not limited to, cancer, diabetes,obesity, diseases of the central nervous system, inflammatory disorders,autoimmune dieseases and cardiovascular diseases. The peptides of thepresent invention also have in vitro utilities, for example, in thegeneration of antibodies which specifically bind the serine/threoninekinase from which the peptide was derived. These antibodies can be usedto identify cells expressing the serine/threonine kinase and to studythe intracellular distribution of the serine/threonine kinase. Inaddition, the peptides of the present invention can be used to identityand quantitate ligands which bind the HJ loop of the serine/threoninekinase from which the peptide was derived.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a sequence illustrating the consensus sequence for amino acidone through amino acid ten of the HJ loop found among the family ofserine/threonine kinases.

FIG. 2 is a sequence illustrating the consensus sequence for amino acidone through amino acid twenty of the HJ loop cyclic AMP dependentprotein kinase and protein kinase C.

FIGS. 3A-3B are a Table illustrating the amino acid sequence of the HJloop of the serine/threonine kinases RAF (SEQ ID NO.: 1), cyclic AMPdependent protein kinase (CAPK) (SEQ ID NO.: 2), protein kinase C (PKC)(SEQ ID NO.: 3), the G-receptor coupled protein kinases β2-adrenergicreceptor kinases 1 and 2 (bARK1.2) (SEQ ID NO.: 4), calmodulin dependentkinase (CaMK) (SEQ ID NO.: 5), polo kinases (SEQ ID NO.: 6), Akt/PKB(SEQ ID NO.: 7) and the G-protein coupled receptor kinases GRK1 (SEQ IDNO.: 8), GRK4 (SEQ ID NO.: 9), GRK5 (SEQ ID NO.: 10), GRK6 (SEQ ID NO.:11) and GSK3 (SEQ ID NO.: 12). Also shown are examples of conservativesubstitutions in these amino acid sequences. An “*” indicates analiphatic, substituted aliphatic, benzylic, substituted benzylic,aromatic or substituted aromatic ester of glutamic acid or asparticacid.

FIG. 4 is a Table illustrating the sequences of the peptides HJ-38 (SEQID NO.: 13), J-41 (SEQ ID NO.: 14), J-42 (SEQ ID NO.: 15), J-43 (SEQ IDNO.: 16), J-43.1 (SEQ ID NO.: 17), J-45 (SEQ ID NO.: 18), J-46 (SEQ IDNO.: 19), J-47 (SEQ ID NO.: 20), J-48 (SEQ ID NO.: 21) and J-29 (SEQ IDNO.: 22). All peptides are N-acetylated and C-amidated. “E!” indicates abenzyl ester of glutamic acid.

DETAILED DESCRIPTION OF THE INVENTION

A serine/threonine kinase (hereinafter “STK”) is an intracellular ormembrane bound protein which uses the gamma phosphate of ATP or GTP togenerate phosphate monoesters on the hydroxyl group of a serine orthreonine residue. STKs have homologous “kinase domains” or “catalyticdomains” which carry out this phosphorylation. Based on a comparison ofa large number of protein kinases, it is now known that the kinasedomain of protein kinases, including STKs, can be divided into twelvesubdomains, which are regions generally uninterrupted by large aminoacid insertions and contain characteristic patterns of conservedresidues (Hanks and Hunter, “The Eukaryotic Protein Kinase Superfamily”,in Hardie and Hanks (ed.), The Protein Kinase Facts Book, Volume I,Academic Press, Chapter 2, 1995. These subdomains are referred to asSubdomain I through Subdomain XII.

The “HJ loop” referred to herein is found within the kinase domain ofSTKs between the middle of Subdomain IX and the middle of Subdomain X.Because of the high degree of homology found in the subdomains ofdifferent protein kinases, including STKs, the amino acid sequences ofthe domains of different STKs can be aligned. Thus, the HJ loop of a STKcan be defined by reference to the amino acid sequence of a prototypicalprotein kinase, for example PKA-Cα, and can be said to correspond to acontiguous sequence of about twenty amino acid residues found betweenabout amino acid 229 and 248 of PKA-Cα.

A second definition of the HJ loop of a STK, which is complementary tothe definition provided in the proceeding paragraph, can be made byreference to the three dimensional structure of the kinase domain ofSTKs. The kinase domain of STKs has been found to contain at least ninealpha helices, referred to as helix A through helix I (Taylor et al.,Phil. Trans. R. Soc. Lond. B340:315 (1993), Mohammadi et al., Cell86:577 (1996) and Hubbard et al., Nature 372:746 (1994)). The HJ loop isa contiguous sequence of about twenty amino acids beginning within the Fhelix about five amino acids residues from the N-terminus of the F helixand extending about five amino acid residues into the G helix.

Optionally, the C-terminus or the N-terminus of the peptides of thepresent invention, or both, can be substituted with a carboxylic acidprotecting group or an amine protecting group, respectively. Suitableprotecting groups are described in Green and Wuts, “Protecting Groups inorganic Synthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, theteachings of which are incorporated herein by reference. Preferredprotecting groups are those which facilitate transport of the peptideinto a cell, for example, by reducing the hydrophilicity and increasingthe lipophilicity of the peptide. Examples of N-terminal protectinggroups include acyl groups (—CO—R₁) and alkoxy carbonyl or aryloxycarbonyl groups (—CO—O—R₁), wherein R₁ is an aliphatic, substitutedaliphatic, benzyl, substituted benzyl, aromatic or a substitutedaromatic group. Specific examples of acyl groups include acetyl,(ethyl)-CO—, n-propyl-CO—, iso-propyl-CO—, n-butyl-CO—, sec-butyl-CO—,t-butyl-CO—, phenyl-CO—, substituted phenyl-CO—, benzyl-CO— and(substituted benzyl)-CO—. Examples of alkoxy carbonyl and aryloxycarbonyl groups include CH₃—O—CO—, (ethyl)-O—CO—, n-propyl-O—CO—,iso-propyl-O—CO—, n-butyl-O—CO—, sec-butyl-O—CO—, t-butyl-O—CO—,phenyl-O—CO—, substituted phenyl-O—CO— and benzyl-O—CO—, (substitutedbenzyl)-O—CO—. The carboxyl group at the C-terminus can be protected,for example, as an an amide (i.e., the hydroxyl group at the C-terminusis replaced with —NH₂, —NHR₂ and —NR₂R₃) or ester (i.e. the hydroxylgroup at the C-terminus is replace with —OR₂). R₂ and R₃ areindependently an aliphatic, substituted aliphatic, benzyl, substitutedbenzyl, aryl or a substituted aryl group. In addition, taken togetherwith the nitrogen atom, R₂ and R₃ can form a C4 to C8 heterocyclic ringwith from about 0-2 additional heteroatoms such as nitrogen, oxygen orsulfur. Examples of suitable heterocyclic rings include piperidinyl,pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples ofC-terminal protecting groups include —NH₂, —NHCH₃, —N(CH₃)₂, —NH(ethyl),—N(ethyl)₂, —N(methyl) (ethyl), —NH(benzyl), —N(C1-C4 alkyl)(benzyl),—NH(phenyl), —N(C1-C4 alkyl) (phenyl), —OCH₃, —O-(ethyl), —O-(n-propyl),—O-(n-butyl), —O-(iso-propyl), —O-(sec-butyl), —O-(t-butyl), —O-benzyland —O-phenyl.

A “peptide derivative of the HJ loop” includes a peptide having theamino acid sequence of the HJ loop. A “peptide derivative of the HJloop” also includes, for example, a subsequence of the HJ loop of theSTK. A subsequence is a contiguous sequence of from about five to abouttwenty amino acids or amino acid residues found within a largersequence. Thus, a subsequence of the HJ loop is a contiguous sequence offrom about five to about twenty amino acids or amino acid residues foundwithin the HJ loop. A subsequence of the HJ loop can also be referred toas a “fragment” of the HJ loop.

A “peptide derivative” also includes a peptide having a “modifiedsequence” in which one or more amino acids in the original sequence orsubsequence have been substituted with a naturally occurring amino acidor amino acid analog (also referred to as a “modified amino acid”). Inone aspect of the present invention, the peptide derivative has ansequence corresponding to a subsequence of the HJ loop of a STK, withthe proviso that any one amino acid residue in the peptide derivativecan differ from the corresponding amino acid residue in the subsequence.For example, if the subsequence is [AA₁]-[AA₂]-[AA₃]-[AA₄]-[AA₅], thenthe peptide derivative can be [AA₁′]-[AA₂]-[AA₃]-[AA₄]-[AA₅],[AA₁]-[AA₂′]-[AA₃]-[AA₄]-[AA₅], [AA₁]-[AA₂]-[AA₃′]-[AA₄]-[AA₅],[AA₁]-[AA₂]-[AA₃]-[AA₄′]-[AA₅] and [AA₁]-[AA₂]-[AA₃]-[AA₄]-[AA5′],wherein [AA′] is a naturally occurring or modified amino acid differentfrom [AA]. In another aspect of the present invention, the peptidederivative has a sequence corresponding to a subsequence of the HJ loopof an STK, with the proviso that any two amino acid residues in thepeptide derivative can differ from the corresponding amino acid residuein the subsequence.

An “amino acid residue” is a moiety found within a peptide and isrepresented by —NH—CHR—CO—, wherein R is the side chain of a naturallyoccurring amino acid. When referring to a moiety found within a peptide,the terms “amino acid residue” and “amino acid” are used interchangeablyin this application. An “amino acid residue analog” includes D or Lresidues having the following formula: —NH—CHR—CO—, wherein R is analiphatic group, a substituted aliphatic group, a benzyl group, asubstituted benzyl group, an aromatic group or a substituted aromaticgroup and wherein R does not correspond to the side chain of anaturally-occurring amino acid. When referring to a moiety found withina peptide, the terms “amino acid residue analog” and “amino acid analog”are used interchangeably in this application.

As used herein, aliphatic groups include straight chained, branched orcyclic C1-C8 hydrocarbons which are completely saturated, which containone or two heteroatoms such as nitrogen, oxygen or sulfur and/or whichcontain one or more units of unsaturation. Aromatic groups includecarbocyclic aromatic groups such as phenyl and naphthyl and heterocyclicaromatic groups such as imidazolyl, indolyl, thienyl, furanyl, pyridyl,pyranyl, pyranyl, oxazolyl, benzothienyl, benzofuranyl, quinolinyl,isoquinolinyl and acridintyl.

Suitable substituents on an aliphatic, aromatic or benzyl group include—OH, halogen (—Br, —Cl, —I and —F) —O(aliphatic, substituted aliphatic,benzyl, substituted benzyl, aryl or substituted aryl group), —CN, —NO₂,—COOH, —NH₂, —NH(aliphatic group, substituted aliphatic, benzyl,substituted benzyl, aryl or substituted aryl group), —N(aliphatic group,substituted aliphatic, benzyl, substituted benzyl, aryl or substitutedaryl group)₂, —COO(aliphatic group, substituted aliphatic, benzyl,substituted benzyl, aryl or substituted aryl group), —CONH₂,—CONH(aliphatic, substituted aliphatic group, benzyl, substitutedbenzyl, aryl or substituted aryl group)), —SH, —S(aliphatic, substitutedaliphatic, benzyl, substituted benzyl, aromatic or substituted aromaticgroup) and —NH—C(═NH)—NH₂. A substituted benzylic or aromatic group canalso have an aliphatic or substituted aliphatic group as a substituent.A substituted aliphatic group can also have a benzyl, substitutedbenzyl, aryl or substituted aryl group as a substituent. A substitutedaliphatic, substituted aromatic or substituted benzyl group can have oneor more substituents.

Suitable substitutions for amino acid residues in the sequence of an HJloop or a subsequence of an HJ loop include conservative substitutionswhich result in peptide derivatives which modulate the activity of aSTK. A “conservative substitution” is a substitution in which thesubstituting amino acid (naturally occurring or modified) has about thesame size and electronic properties as the amino acid being substituted.Thus, the substituting amino acid would have the same or a similarfunctional group in the side chain as the original amino acid.

A “conservative substitution” also refers to utilizing a substitutingamino acid which is identical to the amino acid being substituted exceptthat a functional group in the side chain is functionalized with asuitable protecting group. Suitable protecting groups are described inGreen and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley andSons, Chapters 5 and 7, 1991, the teachings of which are incorporatedherein by reference. As with N-terminal and C-terminal protecting group,preferred protecting groups are those which facilitate transport of thepeptide into a cell, for example, by reducing the hydrophilicity andincreasing the lipophilicity of the peptide, and which can be cleaved invivo, either by hydrolysis or enzymatically, inside the cell. (Ditter etal., J. Pharm. Sci. 57:783 (1968); Ditter et al., J. Pharm. Sci. 57:828(1968); Ditter et al., J. Pharm. Sci. 58:557 (1969); King et al.,Biochemistry 26:2294 (1987); Lindberg et al., Drug Metabolism andDisposition 17:311 (1989); and Tunek et al., Biochem. Pharm. 37:3867(1988), Anderson et al., Arch. Biochem. Biophys. 239:538 (1985) andSinghal et al., FASEB J. 1:220 (1987)). Hydroxyl protecting groupsinclude esters, carbonates and carbamate protecting groups. Amineprotecting groups include alkoxy and aryloxy carbonyl groups, asdescribed above for N-terminal protecting groups. Carboxylic acidprotecting groups include aliphatic, benzylic and aryl esters esters, asdescribed above for C-terminal protecting groups. In one embodiment, thecarboxylic acid group in the side chain of one or more glutamic acid oraspartic acid residue in a peptide of the present invention isprotected, preferably with as a methyl, ethyl, benzyl or substitutedbenzyl ester, more preferably as a benzyl ester.

Provided below are groups of naturally occurring and modified aminoacids in which each amino acid in a group has similar electronic andsteric properties. Thus, a conservative substitution can be made bysubstituting an amino acid with another amino acid from the same group.It is to be understood that these groups are non-limiting, i.e. thatthere are additional modified amino acids which could be included ineach group.

Group I includes leucine, isoleucine, valine, methionine, serine,cysteine, threonine and modified amino acids having the following sidechains: ethyl, n-butyl, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CHOHCH₃ and—CH₂SCH₃. Preferably, Group I includes leucine, isoleucine, valine andmethionine.

Group II includes glycine, alanine, valine, serine, cysteine, threonineand a modified amino acid having an ethyl side chain. Preferably, GroupII includes glycine and alanine.

Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan,cyclohexylmethyl, and modified amino residues having substituted benzylor phenyl side chains. Preferred substituents include one or more of thefollowing: halogen, methyl, ethyl, nitro, methoxy, ethoxy and —CN.Preferably, Group III includes phenylalanine, tyrosine and tryptophan.

Group IV includes glutamic acid, aspartic acid, a substituted orubsubstituted aliphatic, aromatic or benzylic ester of glutamic oraspartic acid (e.g., methyl, ethyl, n-propyl iso-propyl, cyclohexyl,benzyl or substituted benzyl), glutamate, asparagine, CO—NH-alkylatedglutamate or asparagine (e.g., methyl, ethyl, n-propyl and iso-propyl)and modified amino acids having the side chain —(CH₂)₃—COOH, an esterthereof (substituted or unsubstituted aliphatic, aromatic or benzylicester), an amide thereof and a substituted or unsubstituted N-alkylatedamide thereof. Preferably, Group IV includes glutamic acid, asparticacid, methyl aspartate, ethyl asparatate, benzyl asparate and methylglutamate, ethyl glutamate and benzyl glutamate.

Group V includes histidine, lysine, arginine, N-nitroarginine,β-cycloarginine, γ-hydroxyarginine, N-amidinocitruline and2-amino-4-guanidinobutanoic acid, homologs of lysine, homologs ofarginine and ornithine. Preferably, Group V includes histidine, lysine,arginine, and ornithine. A homolog of an amino acid includes from 1 toabout 3 additional methylene units in the side chain.

Group VI includes serine, theronine, cysteine and modified amino acidshaving C1-C5 straight or branched alkyl side chains substituted with —OHor —SH. Preferably, Group VI includes serine, cysteine or threonine.

In another aspect, suitable substitutions for amino acid residues in thesequence of an HJ loop or a subsequence of an HJ loop include “severe”substitutions which result in peptide derivatives which modulate theactivity of a STK. Severe substitutions which result in peptidederivatives that modulate the activity of a STK are much more likely tobe possible in positions which are not highly conserved throughout thefamily of serine/threonine kinases than at positions which are highlyconserved. FIG. 1 shows the consensus sequence of the about first tenamino acids of the HJ loop of STKs. FIG. 2 shows the consensus sequenceof the about twenty amino acids of the HJ loop of cyclic AMP dependentkinase and protein kinase C. Positions which are highly conserved amongthe STK family and the conserved amino acids generally found in thosepositions have been indicated. Positions which are not as highlyconserved among the STK family are indicated with an “X”. BecauseD-amino acids have a hydrogen at a position identical to the glycinehydrogen side-chain, D-amino acids or their analogs can be substitutedfor the glycine at position 6 in FIG. 1 or at positions 6 and 12 in FIG.2.

A “severe substitution” is a substitution in which the substitutingamino acid (naturally occurring or modified) has significantly differentsize and/or electronic properties compared with the amino acid beingsubstituted. Thus, the side chain of the substituting amino acid can besignificantly larger (or smaller) than the side chain of the amino acidbeing substituted and/or can have functional groups with significantlydifferent electronic properties than,the amino acid being substituted.Examples of severe substitutions of this type include the substitutionof phenylalanine or cycohexylmethyl glycine for alanine, isoleucine forglycine, a D amino acid for the corresponding L amino acid or —NH—CH[(—CH₂)₅—COOH]—CO— for aspartic acid. Alternatively, a functional groupmay be added to the side chain, deleted from the side chain or exchangedwith another functional group. Examples of severe substitutions of thistype include adding an amine or hydroxyl, carboxylic acid to thealiphatic side chain of valine, leucine or isoleucine, exchanging thecarboxylic acid in the side chain of aspartic acid or glutamic acid withan amine or deleting the amine group in the side chain of lysine orornithine. In yet another alternative, the side chain of thesubstituting amino acid can have significantly different steric andelectronic properties that the functional group of the amino acid beingsubstituted. Examples of such modifications include tryptophan forglycine, lysine for aspartic acid and —(CH₂)₄COOH for the side chain ofserine. These examples are not meant to be limiting.

Examples STKs whose activity can be modulated by peptide and peptidederivatives, as described herein, include, but are not limited to, STKsbelonging to the following STK families: polo family (Glover et al., J.Cell Biol., 135:1681 (1996)), Raf, mitogen-activated protein kinases(MAP kinases), Akt/PKB (Frank et al., Cell 88:435 (1997) and Hemmings etal., Science 275:628 (1997)) and G protein-coupled receptor kinases.Other suitable STKs include cyclic AMP (cAMP) dependent protein kinase,protein kinase C, calmodulin dependent kinase, glycogen synthasekinase-3 (GSK3) and cyclic GMP (cGMP) dependent protein kinase.

Suitable members of the polo family include, but are not limited to,Plk, Snk and Sak. Suitable members of the Raf family include, but arenot limited to, Raf-1, A-Raf and B-Raf. Suitable G-protein dependentkinases include, but are not limited to, β-adrenergic receptor kinases 1and 2, rhodopsi kinase (GRK1), GRK4, GRK5 and GRK6. Suitable MAP kinasesinclude, but are not limited to MAPK, MAPKK and MAPKKK. Also includedare the protein kinase C isoforms, which include, but are not limitedto, isoforms designated as α, β_(I/H), γ, δ, ε, η(L), Θ, μ, ξ, ι and λ.

The present invention includes peptides having amino acids sequencescorresponding to the sequence found in the HJ loop of STKs, subsequencesthereof and modified subsequences thereof. Examples of suitablesubsequences include, but are not limited to, sequences corresponding to[AA]₁ through [AA]₂₀, [AA]₃ through [AA]₁₀, [AA]₇ through [AA]₁₄, [AA]₁₁through [AA]₁₈, [AA]₃ through [AA]₁₄, [AA]₇ through [AA]₁₈ and [AA]₃through [AA]₁₈ of the HJ loop of a STK, and subsequences thereof. FIGS.3A and 3B show the sequences of the HJ loop of the following STKs: RAF,cyclic AMP dependent kinase, protein kinase C, the G-protein-coupledreceptor kinases βARK 1 and 2 and GRK1, GRK4, GRK5 and GRK6, calmodulindependent kinase, polo, Akt/PKB and GSK3.

FIGS. 3A and 3B also provide a numbering scheme for the amino acidsequence in an loop. The amino acid at the N-terminus of the HJ loop isat position 1 and can be referred to as “[AA]₁”. The next amino acid inthe sequence, referred to as “[AA]₂”, is at position 2 and is followedby amino acids [AA]₃ through [AA]₂₀, which are at positions 3-20. Thus,a peptide 20-mer with an amino acid sequence [AA]₁ through [AA]₂₀includes the twenty amino acids in the HJ loop. A peptide derivative ofthe HJ loop with an amino acid sequence [AA]₃ through [AA]₁₀, as recitedin the preceeding paragraph, includes the third amino acid through thetenth amino acid in said HJ loop.

The present invention also includes peptides having amino acid sequencescorresponding to a modified sequence or subsequence of the HJ loop ofSTKs and which modulate the activity of STKs including RAF, cyclic AMPdependent kinase, protein kinase C, the G-protein-coupled receptorkinases βARK 1, βARK2, GRK1 and GRKs4-6, calmodulin dependent kinase andpolo. In one aspect, one, two or more of the amino acids in the sequenceor subsequence are modified with conservative substitutions; thesubstitutions can be in consensus positions, in non-consensus positionsor in both. In another aspect, one, two or more of the amino acids inthe sequence or subsequence are modified with severe substitutions; thesubstitutions are preferably in non-consensus positions. Also includedare the substitution of conserved glycine residues (e.g., position 6 inFIG. 1 or positions 6 and 12 in FIG. 2) with D-amino acid residues oranalogs thereof. FIGS. 3A and 3B also provide examples of conservativeamino acid substitutions for the HJ loop of RAF, cyclic AMP dependentkinase, protein kinase C, the G-protein-coupled receptor kinases βARK1,βARK2, GRK1 and GRKs4-6, calmodulin dependent kinase, polo, Akt/PKB andGSK3.

Specific examples of peptide derivatives of the present inventioninclude peptides HJ-38 (SEQ ID NO.: 13), J-41 (SEQ ID NO.: 14), J-42(SEQ ID NO.: 15), J-43 (SEQ ID NO.: 16), J-43.1 (SEQ ID NO.: 17), J-45(SEQ ID NO.: 18), J-46 (SEQ ID NO.: 19), J-47 (SEQ ID NO.: 20), J-48(SEQ ID NO.: 21) and J-29 (SEQ ID NO.: 22), the sequences of which areshown in FIG. 4. The N-terminus and/or C-terminus of these peptides canbe modified, as described above. As indicated in FIG. 4, the N-terminalof these peptides is acetylated and the C-terminal is amidated. Otherprotecting groups for amides and carboxylic acids can be used, asdescribed above. Optionally, one or both protecting groups can beomitted. The peptides may be linear or cyclic.

Also included are peptides having the sequence of HJ-38 (SEQ ID NO.:13), J-41 (SEQ ID NO.: 14), J-42 (SEQ ID NO.: 15, J-43 (SEQ ID NO.: 16),J-43.1 (SEQ ID NO.: 17), J-45 (SEQ ID NO.: 18), J-46 (SEQ ID NO.: 19),J-47 (SEQ ID NO.: 20,), J-48 (SEQ ID NO.: 21) and J-29 (SEQ ID NO.: 22),with the proviso that any one of the amino residues in the peptide canvary, being any naturally occurring amino acid or analog thereof. Thepresent invention also includes peptides having the sequence of with theproviso that any two of the amino residues in the peptide can vary,being any naturally occurring amino acid or analog thereof.

The present invention also includes cyclic peptides having amino acidssequences corresponding to a modified sequence or subsequence of the HJloop of STKs and which modulate the activity of STKs.

A “cyclic peptide” refers, for example, to a peptide or peptidederivative in which a ring is formed by a peptide bond between thenitrogen atom at the N-terminus and the carbonyl carbon at theC-terminus.

“Cyclized” also refers to forming a ring by a covalent bond between thenitrogen at the N-terminus of the compound and the side chain of asuitable amino acid in the peptide, preferably the C-terminal aminoacid. For example, an amide can be formed between the nitrogen atom atthe N-terminus and the carbonyl carbon in the side chain of asparticacid or glutamic acid. Alternatively, the peptide or peptide derivativecan be cyclized by forming a covalent bond between the carbonyl at theC-terminus of the compound and the side chain of a suitable amino acidin the peptide, preferably the N-terminal amino acid. For example, anamide can be formed between the carbonyl carbon at the C-terminus andthe amino nitrogen atom in the side chain of lysine or ornithine; anester can be formed between the carbonyl carbon at the C-terminus andthe hydroxyl oxygen atom in the side chain of serine or threonine.

“Cyclized” also refers to forming a ring by a covalent bond between theside chains of two suitable amino acids in the peptide, preferably theterminal amino acids. For example, a disulfide can be formed between thesulfur atoms in the side chains of two cysteines. Alternatively, anester can be formed between the carbonyl carbon in the side chain of,for example, glutamic acid or aspartic acid, and the oxygen atom in theside chain of, for example, serine or threonine. An amide can be formedbetween the carbonyl carbon in the side chain of, for example, glutamicacid or aspartic acid, and the amino nitrogen in side chain of, forexample, lysine or ornithine.

In addition, a peptide or peptide derivative can be cyclized with alinking group between the two termini, between one terminus and the sidechain of an amino acid in the peptide or peptide derivative, or betweenthe side chains to two amino acids in the peptide or peptide derivative.Suitable linking groups are disclosed in Lobl et al., WO 92/00995 andChiang et al., WO 94/15958, the teachings of which are incorporated intothis application by reference.

Suitable substitutions in the original amino acid sequence orsubsequence are those which result in a peptide derivative, as definedabove, which modulates the activity of a STK. The activity of a STK is“modulated” when the activity of the STK is increased or decreased. Anincrease or decrease in the activity of a STK can be detected byassessing in vitro the extent of phosphorylation of a protein substrateof the STK being tested or by a corresponding modulation, increase ordecrease, in a cellular activity or function which is under the controlof the STK. Examples of these cellular functions include cellproliferation, cell differentiation, cell morphology, cell survival orapoptosis, cell response to external stimuli, gene expression, lipidmetabolism, glycogen metabolism and mitosis.

It can be readily determined whether a peptide or peptide derivativemodulates the activity of a STK by providing cells which have one ormore cellular activities controlled by a STK. The cells are incubatedwith the peptide or peptide derivative to produce a test mixture underconditions suitable for assessing activity of the serine/threoninekinase. The activity of the STK is assessed and compared with a suitablecontrol, e.g., the activity of the same cells incubated under the sameconditions in the absence of the peptide or peptide derivative. Agreater or lesser activity of the STK in the test mixture compared withthe control indicates that the test peptide or peptide derivativemodulates the activity of said STK.

Suitable cells for the assay include normal cells which express amembrane bound or intracellular STK, cells which have been geneticallyengineered to express a STK, malignant cells expressing a STK orimmortalized cells which express a STK.

Conditions suitable for assessing STK activity include conditionssuitable for assessing activity of a cellular activity or function undercontrol of the STK. Generally, a cellular activity or function can beassessed when the cells are exposed to conditions suitable for cellgrowth, including a suitable temperature (for example, between about 30°C. to about 42° C.) and the presence of the suitable concentrations ofnutrients in the medium (e.g., amino acids, vitamins, growth factors).

In another aspect, the activity of certain STK (e.g., Atk/PKB, Dudek etal., Science 275:661 (1997)) can be evaluated by growing the cells underserum deprivation conditions. Cells are typically grown in culture inthe presence of a serum such as bovine serum, horse serum or fetal calfserum. Many cells, for example, nerve cells such as PC-12 cells,generally do not survive when insufficient serum. The use ofinsufficient serum to culture cells is referred to as “serum deprivationconditions” and includes, for example, from 0% to about 4% serum. STKactivity is determined by the extent to which a peptide or peptidederivative can protect cells, e.g., neuronal cells, from theconsequences of serum deprivation. Specific conditions are provided inDudek et al., and in Example 4 of co-pending and concurrently filedapplication entitled “SHORT PEPTIDES WHICH SELECTIVELY MODULATEINTRACELLULAR SIGNALLING” (filed on May 21, 1997, Attorney Docket No.CMCC-547), the teachings of which are incorporated herein by reference.

Generally, the activity of the STK in the test mixture is assessed bymaking a quantitative measure of the cellular activity which the STKcontrols. The cellular activity can be, for example, cell proliferation.Examples of cells in which proliferation is controlled by an STK includeendothelial cells such as bovine aortic cells, mouse MSI cells or mouseSVR cells (see Arbiser et al., Proc. Natl. Acad. Sci. USA 94:861(1997)), vascular smooth muscle cells, and malignant cells of varioustissues such as breast cancer, lung cancer, colon cancer, prostratecancer, melanoma). STK activity is assessed by measuring cellularproliferation, for example, by comparing the number cells present aftera given period of time with the number of cells originally present. STKsinvolved in cell proliferation are members of the polo family, Taf orAtk/PKB. If cells are being used in which the STK controls the celldifferentitiation (e.g., preadipocytes such as 3T3-L1 expressing STKsAkt/PKB, GSK3 and protein kinase A—see Kohn et al., J. Biol. Chem.271:31372 (1996)), activity is assessed by measuring the degree ofdifferentiation. Activity can be assessed by changes in the metabolicactivity of cells such as primary adipocytes, hepatocytes andfibroblasts by measuring changes in glucose uptake, lipogenesis, orglycogen metabolism (see, for example, Weise et al., J. Biol. Chem.270:3442 (1995)). Activity can also be assessed by the extent to whichthe gene expression, cell morphology or cellular phenotype is altered(e.g., the degree to which cell shape is altered or the degree to whichthe cells assume a spindle-like structure). One example of a change incellular morphology is reported in the co-pending and concurrently filedapplication entitled “SHORT PEPTIDES WHICH SELECTIVELY MODULATEINTRACELLULAR SIGNALLING” (filed on May 21, 1997, Attorney Docket No.CMCC-547), which discloses that certain peptide derivatives of the HJloop of protein tyrosine kinases can cause vascular smooth muscle cellsto become elongated and assume a spindle-like shape.

Specific examples of conditions suitable for determining the activity ofSTKs by assessing cell proliferation are provided in Example 2.

It is to be understood that the assay described hereinabove fordetermining whether a peptide or peptide derivative modulates a cellularactivity or function under the control of a STK can be performed withcells other than those specifically described herein. STKs not yetdiscovered or STKs whose function is not yet known can also be used inthis assay, once it has been determined which cellular functions oractivities they control. These STKs are also within the scope of thepresent invention.

The present invention is also directed to a method of modulating theactivity of a serine/threonine kinase in a subject. A “subject” ispreferably a human, but can also be animals in need of treatment, e.g.,veterinary animals (e.g., dogs, cats, and the like), farm animals (e.g.,cows, pigs, horses and the like) and laboratory animals (e.g., rats,mice, guinea pigs and the like).

The activity of a STK in a subject can be modulated for the purpose oftreating diseases which are caused by over activity or under activity ofSTKs. For example, MAP kinases (Seger and Krebs, FASEB J. 9:726 (1995))and cyclin dependent protein kinases (“Molecular Biology of the Cell,”Alberts, Bray, Lewis, Raff, Roberts and Watson, eds. Chapter 5, (GarlandPublishing, Inc.), (1994)), are central components of the cell-divisioncycle control system in eukaryotic cells. Other STKs, for example,protein kinase C, Raf kinases (Nishizuka, The FASEB Journal 9:484(1995), Locric, et al., Oncogene 12:1109 (1996) and Laird et al., J.Siol. Chem. 270:26,742 (1995)) and G protein-coupled receptors(Lange-Carter, et al., Science 260:315 (1993)), are, in turn, involvedin the control of MAP kinases or are activated during mitosis. The Gprotein-coupled receptor kinases (GRKs), on the other hand, desensitizethe receptors and are thereby involved in the regulation of varioushormonal responses (Freedman and Lefkowitz, Recent Prog. Hormon. Res.51:319 (1996). Activation of Akt/PKB is implicated in the inhibition ofapoptosis, i.e., programmed cell death (Frank et al., Cell 88:435 (1997)and Hemmings Science 275:628 (1997)). Peptides and peptide derivativesof the present invention which modulate the activity of these enzymescan be used to treat cancer in a subject when administered to thesubject in a therapeutically effective amount.

c-AMP dependent kinase, GSK3 and Akt/PKB are involved in the control ofglycogen metabolism. Peptide and peptide derivatives of the presentinvention which modulate the activity of cAMP dependent kinase can beused to treat Type II diabetes and hemorrhagic shock in a subject whenadministered to the subject in a therapeutically effective amount. cAMPderivatives have also been reported to inhibit the growth of humancancer cells (Katsros et al., FEBS Lett. 223:97 (1987)), indicating thatinhibitors of cAMP dependent kinases can also be useful in the treatmentof cancer.

Raf kinases are involved in the control of lipid metabolism. Peptide andpeptide derivatives of the present invention which modulate the activityof Raf kinases can be used to treat obesity in a subject whenadministered to the subject in a therapeutically effective amount.

Agents which modulate the activity of protein kinase C can be used totreat a wide variety of disease conditions, including cardiovasculardiseases (e.g., thrombosis, atherosclerosis, arteriosclerosis, cardiachypertrophy, ischemia, reperfusion injury and hypertension),immunosuppresive and inflammatory disorders (e.g., asthma, psoriasis,systemic lupus erythematosus, diabetes mellitus, supression of organtransplant rejection, multiple sclerosis, inflammatory bowel disease andAIDS), central nervous system diseases (e.g., Alzheimer's disease,stroke and trauma), septic shock based on protein kinase C activationand ischemia induced renal failure (Nambi, WO 93/16703, Bradshaw, etal., Agents Action 38:135 (1993) and Birchall et al., The J. Pharm. andExper. Therapeut. 2:922 (1994)). Peptide and peptide derivatives of thepresent invention which modulate the activity of protein kinase C can beused to treat these diseases in a subject when administered to thesubject in a therapeutically effective amount.

Phosphorylation by G-protein receptor kinases are known (Freedman andLefkowitz, Recent Prog. Hormon. Res. 51:319 (1996)) to result inreceptor desensitization, thereby extending the extending the durationof hormonal effects of, for example, adrenalin. Thus, agents whichmodulate the activity of G-protein receptor kinases have potential inthe treatment of disease resulting from a lower bioavailability of thecorresponding ligand, such as dopamine. Inhibitors of calmodulindependent kinases have been reported to inhibit dopamine release(Nagatsu et al., Biochem. Biophys. Research, Commun. 143:1045 (1987)).Thus, agents which modulate the activity of G-protein receptor kinasesand calmodulin receptor kinases are potentially useful in the treatmentof diseases involving dysfunction of dopamine signalling, for example,Parkinson's Disease. Inhibitors of calmodulin dependent kinases havealso been reported to relax arterial muscle (Saitoh et al., J. Bio.Chem. 262:7796 (1987)) and therefore have potential in treatinghypertension. Inhibition of GSK3 might increase the intracellularactivity of the insulin receptor and thereby enhance glucose uptake andother related metabolic activities. Thus, agents which modulate theactivity of GSK3 are potentially useful in the treatment of Type I andType II diabetes.

Based on methods disclosed herein, peptides and peptide derivatives canbe designed to modulate the activity of STKs whose HJ loop has beensequenced and whose cellular function is known. As a consequence,peptides and peptide derivatives can be designed to affect (increase ordecrease) those cellular functions. It is possible that future researchwill reveal that certain disease conditions, whose underlying causes arepresently unknown, are brought about by the overactivity orunderactivity of cellular functions controlled by STKs. These diseasescan be treated by administering peptides which are peptide derivativesof the HJ loop of the overactive or underactive STK. Suitable peptidesand peptide derivatives can be identified by methods disclosed herein.These methods of treatment, peptides and peptide derivatives areencompassed within the scope of the present invention.

A “therapeutically effective amount” is the quantity of compound whichresults in an improved clinical outcome as a result of the treatmentcompared with a typical clinical outcome in the absence of thetreatment. An “improved clinical outcome” includes a longer lifeexpectancy for individuals with the disease as a result of thetreatment. An “improved clinical outcome” can also result in theindividual with the disease experiencing fewer symptoms or complicationsof the disease as a result of the treatment. With respect to cancer, an“improved clinical outcome” includes a longer life expectancy. It canalso include slowing or arresting the rate of growth of a tumor, causinga shrinkage in the size of the tumor, a decreased rate of metastasisand/or improved quality of life (e.g., a decrease in physical discomfortor an increase in mobility).

With respect to diabetes, an improved clinical outcome refers to alonger life expectancy, a reduction in the complications of the disease(e.g., neuropathy, retinopathy, nephropathy and degeneration of bloodvessels) and an improved quality of life, as described above.

With respect to obesity, an improved clinical outcome refers toincreased weight reduction per calory intake. It also refers to adecrease in the complications which are a consequence of obesity, forexample heart disease such as arteriosclerosis and high blood pressure.

The amount of peptide or peptide derivative administered to theindividual will depend on the type and severity of the disease and onthe characteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors.Typically, a therapeutically effective amount of the peptide or peptidederivative can range from about 1 mg per day to about 1000 mg per dayfor an adult. Preferably, the dosage ranges from about 1 mg per day toabout 100 mg per day.

The peptide and peptide derivatives of the present invention arepreferably administered parenterally. Parenteral administration caninclude, for example, systemic administration, such as by intramuscular,intravenous, subcutaneous, or intraperitoneal injection. Peptides orpeptide derivatives which resist proteolysis can be administered orally,for example, in capsules, suspensions or tablets.

The peptide or peptide derivative can be administered to the individualin conjunction with an acceptable pharmaceutical carrier as part of apharmaceutical composition for treating the diseases discussed above.Suitable pharmaceutical carriers may contain inert ingredients which donot interact with the peptide or peptide derivative. Standardpharmaceutical formulation techniques may be employed such as thosedescribed in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa. Suitable pharmaceutical carriers for parenteraladministration include, for example, sterile water, physiologicalsaline, bacteriostatic saline (saline containing about 0.9% mg/ml benzylalcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactateand the like. Methods for encapsulating compositions (such as in acoating of hard gelatin or cyclodextran) are known in the art (Baker, etal., Controlled Release of Biological Active Agents, John Wiley andSons, 1986).

The peptide and peptide derivatives of the present invention have manyutilities other than for therapy. Some of these uses are discussed inthe following paragraphs.

The HJ loop peptides of the present invention are derived from an arraywhich is linear in the native protein. Therefore, they can be useful inthe preparation of specific antibodies against STKs. Moreover, since theHJ-loop sequence is unique to each sub-family of STK, anti-HJ-loopantibodies can be specifically used to isolate distinct sub-families ofSTK.

Suitable antibodies can be raised against an HJ loop peptide byconjugating to a suitable carrier, such as keyhole limpet hemocyanin orserum albumin; polyclonal and monoclonal antibody production can beperformed using any suitable technique. A variety of methods have beendescribed (see e.g., Kohler et al., Nature, 256: 495-497 (1975) and Eur.J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266: 550-552(1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D.Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.); Current Protocols In MolecularBiology, Vol. 2 (Supplement 27, Summer 1994), Ausubel, F. M. et al.,Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)).Generally, a hybridoma can be produced by fusing a suitable immortalcell line (e.g., a myeloma cell line such as SP2/0) with antibodyproducing cells. The antibody producing cell, preferably those of thespleen or lymph nodes, can be obtained from animals immunized with theantigen of interest. The fused cells (hybridomas) can be isolated usingselective culture conditions, and cloned by limiting dilution. Cellswhich produce antibodies with the desired specificity can be selected bya suitable assay (e.g., ELISA).

Antibodies, including monoclonal antibodies, against HJ loop peptideshave a variety of uses. For example, those against or reactive with theprotein from which the HJ peptides was derived, and preferably whichbind specifically to said protein, can be used to identify and/or sortcells exhibiting that protein on the cell surface (e.g., by means offlourescence activated cell sorting or histological analyses).Monoclonal antibodies specific for the protein can also be used todetect and/or quantitate the protein expressed on the surface of a cellor present in a sample (e.g., in an ELISA). Alternatively, theantibodies can be used to determine if an intracellular STK is presentin the cytoplasm of the cell. A cleared lysate of the cell is generated(for example, by treating the cells with sodium hydroxide (0.2 N) andsodium dodecyl sulfate (1%), centrifugating and separating thesupernatant from the pellet), and treated with anti-HJ loop antibodyspecific for the STK. The cleared lysate is then analzyed, for example,by Western blotting or immunoprecipitation for complexes between STK andantibody. Some STKs become membrane-bound or cytoskeleton-associatedfollowing stimulation. Anti-HJ-loop antibodies can be utilized for thestudy of the intracellular distribution (compartmentalization) ofvarious subfamilies of STKs under various physiologigal conditions viathe application of conventional immunocytochemistry such asimmunofluoresence, immunoperoxidase technique and immunoelectronmicroscopy, in conjunction with the specific anti-HJ-loop antibody.

Antibodies reactive with the immunogen are also useful. For example,they can be used to detect and/or quantitate immunogen in a sample, orto purify immunogen (e.g., by immunoaffinity purification).

The HJ loop within STKs plays a key role in regulating the activity ofSTKs, as is evidenced by the fact that the peptides and peptidederivatives of the present invention have such a dramatic effect on theactivity of STKs. The HJ loop peptides of the present invention can alsobe used to identify ligands which interact with the HJ-loops of specificSTKs and which modulate the activity STKs. For example, an affinitycolumn can be prepared to which a specific HJ-loop is covalentlyattached, directly or via a linker. This column, in turn, can beutilized for the isolation and identification of specific ligands whichbind the HJ loop peptide and which will also likely bind the STK fromwhich the HJ loop peptide was derived. The ligand can then be elutedfrom the column, characterized and tested for its ability modulate STKfunction.

Protein tyrosine kinases are another class of protein kinases. Theseproteins occur as membrane-bound receptors, which participate intransmembrane signaling, or as intracellular proteins which take part insignal transduction within the cell, including signal transduction tothe nucleus. Binding of a ligand results in signal transduction,initiated by the phosphorylation of tyrosine residues of intracellularproteins by the kinase. As with STKs, tyrosine kinases control cellularfunctions by means of this phosphorylation mechanism. Tyrosine kinaeshave a high degree of homology with STKs, including an HJ loop.Consequently, the activity of tyrosine kinases and the cellularfunctions which they control, can be modulated with peptides which arepeptide derivatives of their HJ loops, as discussed above for STKs.Peptides and peptides derivatives of the HJ loop of protein tyrosinekinases and methods of use thereof are disclosed in the co-pending andconcurrently filed application entitled “SHORT PEPTIDES WHICHSELECTIVELY MODULATE INTRACELLULAR SIGNALLING” (Attorney Docket No.CMCC-547, filed May 21, 1997), the teachings of which are incorporatedinto this application.

Peptide sequences in the compounds of the present invention may besynthesized by solid phase peptide synthesis (e.g., BOC or FMOC) method,by solution phase synthesis, or by other suitable techniques includingcombinations of the foregoing methods. The BOC and FMOC methods, whichare established and widely used, are described in Merrifield, J. Am.Chem. Soc. 88:2149 (1963); Meienhofer, Hormonal Proteins and Peptides,C. H. Li, Ed., Academic Press, 1983, pp. 48-267; and Barany andMerrifield, in The Peptides, E. Gross and J. Meienhofer, Eds., AcademicPress, New York, 1980, pp. 3-285. Methods of solid phase peptidesynthesis are described in Merrifield, R. B., Science, 232: 341 (1986);Carpino, L. A. and Han, G. Y., J. Org. Chem., 37: 3404 (1972); andGauspohl, H. et al., Synthesis, 5: 315 (1992)). The teachings of thesereferences are incorporated herein by reference.

Methods of cyclizing compounds having peptide sequences are described,for example, in Lob1 et al., WO 92/00995, the teachings of which areincorporated herein by reference. Cyclized compounds can be prepared byprotecting the side chains of the two amino acids to be used in the ringclosure with groups that can be selectively removed while all otherside-chain protecting groups remain intact. Selective deprotection isbest achieved by using orthogonal side-chain protection such as allyl(OAI) (for the carboxyl group in the side chain of glutamic acid oraspartic acid, for example), allyloxy carbonyl (Aloc) (for the aminonitrogen in the side chain of lysine or ornithine, for example) oracetamidomethyl (Acm) (for the sulfhydryl of cysteine) protectinggroups. OAI and Aloc are easily removed by Pd^(o) and Acm is easilyremoved by iodine treatment.

The invention is illustrated by the following examples which are notintended to be limiting in any way.

EXAMPLE 1

Preparation of HJ Peptides

The novel compounds of this invention can be synthesized utilizing a430A Peptide Synthesizer from Applied Biosystems using F-Moc technologyaccording to manufacturer's protocols. Other suitable methodologies forpreparing peptides are known to person skilled in the art. See e.g.,Merrifield, R. B., Science, 232: 341 (1986); Carpino, L. A., Han, G. Y.,J. Org. Chem., 37: 3404 (1972); Gauspohl, H., et al., Synthesis, 5: 315(1992)), the teachings of which are incorporated herein by reference.

Rink Amide Resin [4(2′,4′ Dimethoxyphenyl-FMOC amino methyl) phenoxyresin] was used for the synthesis of C-amidated peptides. Thealpha-amino group of the amino acid was protected by an FMOC group,which was removed at the beginning of each cycle by a weak base, 20%piperidine in N-methylpyrrolidone (NMP). After deprotection, the resinwas washed with NMP to remove the piperidine. In situ activation of theamino acid derivative was performed by the FASTMOC Chemistry using HBTU(2(1-benzotriazolyl-1-yl)-1,1,3,3-tetramethyluronium) dissolved in HOBt(1-hydroxybenzotriazole) and DMF (dimethylformamide). The amino acid wasdissolved in this solution with additional NMP. DIEA(diisopropylethylamine) was added to initiate activation. Alternatively,the activation method of DCC (dicyclohexylcarbodiimide) and HOBt wasutilized to form an HOBt active ester. Coupling was performed in NMP.Following acetylation of the N-terminus (optional), TFA (trifluoroaceticacid) cleavage procedure of the peptide from the resin and the sidechain protecting groups was applied using 0.75 g crystalline phenol;0.25 ml EDT (1,2-ethandithiol); 0.5 ml thioanisole; 0.5 ml D.I. H₂O; 10ml TFA.

EXAMPLE 2

HJ Peptide Derivatives of Raf and Polo Modulate Proliferation ofEndothelial Cells In Vitro

Bovine aortic cells (referred to herein as “A19 cells”) were obtained bythe procedure disclosed in Gospodorowicz et al., Proc. Natl. Acad. Sci.73:4120 (1976)). Mouse MS1 and SVR cells were obtained by the proceduresdisclosed in Arbiser et al., Proc. Natl. Acad. Sci. 94:861 (1997), theteachings of which are incorporated herein by reference.

96 well, flat bottom, tissue culture microtiter plates were precoatedwith gelatin (Difco) immediately prior to cell plating by adding 0.100ml/well of freshly filtered 1% gelatin in glass double distilled water(DDW). The wells were incubated for about 1 hour at 37° C., and then theexcess solution was removed by aspiration.

Culture medium was prepared from DMEM, pencillin/streptomycin/glutamine(penicillin—100 U/ml; streptomycin—100 μg/mL; and glutamine—2 mM) and10% endotoxin free bovine calf serum (Hyclone). A suspension of the celltype being tested at 25×10³ cells/ml was prepared in the above describedculture medium and distributed 0.160 ml/well (about 4000 endothelialcells/well).

A series of HJ peptide stock solutions was prepared by diluting a 10 mMsolution of the HJ peptide in 100% DMSO with phosphate buffered saline(PBS)containing 0.1% BSA. The concentration of HJ peptide in each stocksolution was adjusted to nine times the desired concentration of the HJpeptide in the assay mixture.

0.020 ml of each HJ peptide stock solution was added to thecorresponding wells about 2 hours after cell plating, with sixreplicates for each concentration. In addition, BSA solution with noadded HJ peptide was used as a control. The wells were incubated for72-80 hours at 37° C. in a 10% CO₂ humidified incubator.

The plates were labeled and the medium discarded. Each plate was thenwashed one time with PBS (0.200 ml/well). The wells were then fixed bywashing with 100% ethanol (0.200 ml/well for 5 minutes). The ethanol wasremoved and the wells dried completely. Alternatively, the wells werefixed with 4% formaldehyde PBS (PBS buffered 10% formalin from FisherScientific; Catalog No. HC200-1) (0.12 ml/well) for at least 30 minutes.Fixing with formaldehyde enhances the O.D. compared with ethanol.

The wells were washed one time with borate buffer (0.1 M, pH 8.5).Freshly filtered 1% methylene blue solution (0.600 ml/well) was thenadded to the wells and incubated for 10 minutes at room temperature. Thewells were then washed five times with tap water, after which the wellswere dried completely. 0.200 ml/well of 0.1 N HCl (0.1 N) was added toextract the color. After extracting overnight, the O.D. was read at 630nm to determine the number of cells per well. The procedure for countingcells is described in greater detail in Oliver et al., J. of Cell Sci.,92:513 (1989), the teachings of which are incorporated herein byreference.

The results for a number of different HJ peptides are shown in theTable.

TABLE S.I.* (μM) S.I.* (μM) S.I.* (μM) Peptide for SVR Cells for MS1Cells for A19 Cells HJ38 10 10 Not Tested J41 Not Tested 10 Not TestedJ42 10 Not Tested 10 J43 Not Tested Not Tested 40 *Concentration atwhich significant inhibition of cell proliferation was observed.

As can be can from the results in the Table, HJ peptide derivatives ofRaf and Polo inhibited cell proliferation of bovine aortic cells and thetransformed mouse cell lines MS1 and SVR.

Equivalents

Those skilled in the art will be able to recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

22 20 amino acids amino acid Not Relevant Not Relevant peptide unknown 1Tyr Glu Leu Met Thr Gly Glu Leu Pro Tyr Ser His Ile Asn Asn Arg 1 5 1015 Asp Gln Ile Ile 20 20 amino acids amino acid Not Relevant NotRelevant peptide unknown 2 Tyr Glu Met Ala Ala Gly Tyr Pro Pro Phe PheAla Asp Gln Pro Ile 1 5 10 15 Gln Ile Tyr Glu 20 20 amino acids aminoacid Not Relevant Not Relevant peptide unknown 3 Tyr Glu Met Leu Ala GlyGln Pro Pro Phe Asp Gly Glu Asp Glu Asp 1 5 10 15 Glu Leu Phe Gln 20 20amino acids amino acid Not Relevant Not Relevant peptide unknown 4 PheLys Leu Ile Arg Gly His Ser Pro Phe Arg Gln His Lys Thr Lys 1 5 10 15Asp Lys His Glu 20 20 amino acids amino acid Not Relevant Not Relevantpeptide unknown 5 Tyr Ile Leu Leu Val Gly Tyr Pro Pro Phe Trp Asp GluAsp Gln His 1 5 10 15 Arg Leu Tyr Gln 20 20 amino acids amino acid NotRelevant Not Relevant peptide unknown 6 Tyr Thr Leu Leu Val Gly Lys ProPro Phe Glu Thr Ser Cys Leu Lys 1 5 10 15 Glu Thr Tyr Leu 20 20 aminoacids amino acid Not Relevant Not Relevant peptide unknown 7 Tyr Glu MetMet Cys Gly Arg Leu Pro Phe Tyr Asn Gln Asp His Glu 1 5 10 15 Arg LeuPhe Glu 20 20 amino acids amino acid Not Relevant Not Relevant peptideunknown 8 Tyr Glu Met Ile Ala Ala Arg Gly Pro Phe Arg Ala Arg Gly GluLys 1 5 10 15 Val Glu Asn Lys 20 20 amino acids amino acid Not RelevantNot Relevant peptide unknown 9 Tyr Glu Met Ile Gln Gly His Ser Pro PheLys Lys Tyr Lys Glu Lys 1 5 10 15 Val Lys Trp Glu 20 20 amino acidsamino acid Not Relevant Not Relevant peptide unknown 10 Tyr Glu Met IleGlu Gly Gln Ser Pro Phe Arg Gly Arg Lys Glu Lys 1 5 10 15 Val Lys ArgGlu 20 20 amino acids amino acid Not Relevant Not Relevant peptideunknown 11 Tyr Glu Met Ile Ala Gly Gln Ser Pro Phe Gln Gln Arg Lys LysLys 1 5 10 15 Ile Lys Arg Glu 20 20 amino acids amino acid Not RelevantNot Relevant peptide unknown 12 Ala Glu Leu Leu Leu Gly Gln Pro Ile PhePro Gly Asp Ser Gly Val 1 5 10 15 Asp Gln Leu Val 20 8 amino acids aminoacid Not Relevant Not Relevant peptide unknown Modified-site /note=“N-Acetyl Valine” Modified-site /note= “Phenylalanine-NH2” 13 Val MetThr Gly Gln Leu Pro Phe 1 5 8 amino acids amino acid Not Relevant NotRelevant peptide unknown Modified-site /note= “N-Acetyl Valine”Modified-site /note= “Glutamic Acid Benzyl Ester” Modified-site /note=“Phenylalanine-NH2” 14 Val Met Thr Gly Glu Leu Pro Phe 1 5 9 amino acidsamino acid Not Relevant Not Relevant peptide unknown Modified-site/note= “N-Acetyl Methionine” Modified-site /note= “Gamma Benzyl Ester ofGlutamic Acid-NH2” 15 Met Leu Leu Gly Arg Pro Pro Phe Glu 1 5 8 aminoacids amino acid Not Relevant Not Relevant peptide unknown Modified-site/note= “N-Acetyl Methionine” Modified-site /note= “Phenylalanine-NH2” 16Met Leu Leu Gly Lys Pro Pro Phe 1 5 9 amino acids amino acid NotRelevant Not Relevant peptide unknown Modified-site /note= “N-AcetylMethionine” Modified-site /note= “Gamma Benzyl Ester of GlutamineAcid-NH2” 17 Met Leu Leu Gly Lys Pro Pro Phe Glu 1 5 9 amino acids aminoacid Not Relevant Not Relevant peptide unknown Modified-site /note=“N-Acetyl Leucine” Modified-site /note= “Glutamic Acid Benzyl Ester”Modified-site /note= “Serine-NH2” 18 Leu Gly Arg Pro Pro Phe Glu Thr Ser1 5 11 amino acids amino acid Not Relevant Not Relevant peptide unknownModified-site /note= “N-Acetyl Methionine” Modified-site /note=“Glutamic Acid Benzyl Ester” Modified-site 11 /note= “Serine-NH2” 19 MetLeu Leu Gly Arg Pro Pro Phe Glu Thr Ser 1 5 10 7 amino acids amino acidNot Relevant Not Relevant peptide unknown Modified-site /note= “N-AcetylGlycine” Modified-site /note= “Asparagine-NH2” 20 Gly Arg Leu Pro PhePhe Asn 1 5 11 amino acids amino acid Not Relevant Not Relevant peptideunknown Modified-site /note= “N-Acetyl Glutamic Acid Benzyl Ester”Modified-site 11 /note= “Asparagine-NH2” 21 Glu Met Met Ser Gly Arg LeuPro Phe Phe Asn 1 5 10 10 amino acids amino acid Not Relevant NotRelevant peptide unknown Modified-site /note= “N-Acetyl Leucine”Modified-site 10 /note= “Glycine-NH2” 22 Leu Leu Leu Gly Gln Pro Ile PhePro Gly 1 5 10

What is claimed is:
 1. A peptide comprising a peptide derivative of theHJ loop of a serine/threonine kinase, wherein: a) said peptide hasbetween about five and about twenty amino acids or amino acid analogs;and b) said peptide modulates activity of the serine/threonine kinase.2. The peptide of claim 1 wherein the peptide is linear.
 3. The peptideof claim 2 wherein either or both the N-terminus or the C-terminus ofsaid peptide is substituted.
 4. The peptide of claim 3 wherein theN-terminus is amidated and the C-terminus is acylated.
 5. The peptide ofclaim 2 wherein the serine/threonine kinase is a member of the poloserine/threonine kinase family.
 6. The peptide of claim 5 wherein theserine/threonine kinase is a member of the polo family and is selectedfrom the group consisting of Plk, Snk and Sak.
 7. The peptide of claim 2wherein the peptide is represented by a sequence or a subsequence ofsaid HJ loop, wherein the subsequence has at least about five aminoacids.
 8. The peptide of claim 2 wherein the peptide has the sequence ofJ-42 (SEQ ID NO.: 15), J-43 (SEQ ID NO.: 16), J-43.1 (SEQ ID NO.: 17),J-45 (SEQ ID NO.: 18) or J-46 (SEQ ID NO.: 19).
 9. A peptide consistingof about twenty amino acids, AA₁ through AA₂₀, or a subsequence thereofcomprising at least five amino acids, wherein: AA₁ is selected from thegroup consisting of tyrosine, phenylalanine and tryptophan; AA₂ isselected from the group consisting of serine and threonine; AA₃ isselected from the group consisting of leucine, isoleucine, methionineand valine; AA₄ is selected from the group consisting of leucine,isoleucine, methionine and valine; AA₅ is selected from the groupconsisting of leucine, isoleucine, methionine and valine; AA₆ is glycineor alanine; AA₇ is selected from the group consisting of arginine,N-nitroarginine, β-cycloarginine, γ-hydroxyarginine, amidinocitruline,2-amino-4-guanidinobutanoic acid lysine and ornithine; AA₈ is proline;AA₉ is proline; AA₁₀ is selected from the group consisting of tyrosine,phenylalanine and tryptophan; AA₁₁ is selected from the group consistingof asparagine, glutamine, glutamic acid, aspartic acid and an aliphatic,substituted aliphatic, benzyl, substituted benzyl, aromatic orsubstituted aromatic ester of glutamic acid or aspartic acid; AA₁₂ isserine or threonine; AA₁₃ is serine or threonine; AA₁₄ is selected fromthe group consisting of cysteine, serine and threonine; AA₁₅ is selectedfrom the group consisting of leucine, isoleucine, methionine and valine;AA₁₆ is lysine or ornithine; AA₁₇ is selected from the group consistingof glutamine, asparagine, glutamic acid, aspartic acid and an aliphatic,substituted aliphatic, benzyl, substituted benzyl, aromatic orsubstituted aromatic ester of glutamic acid or aspartic acid; AA₁₈ isserine or threonine; AA₁₉ is selected from the group consisting oftyrosine, phenylalanine and tryptophan; and AA₂₀ is selected from thegroup consisting of leucine, isoleucine, methionine and valine.